1. Choose a polymerase that matches your need
2. Take time to carefully to design your primers
Primer design
l 20-30 nucleotides
l GC content 40-60%
l Tms witnin with 5 degrees of each other
3.Calculate annealing temperature
*Always recalculate annealing temperature when switching enzymes
4.Calculate GC content of your target
5.Setup on ice and preheat thermoclcler
Medicalet Centrifuge tube with
l Room temperature reaction setup
l Increased specificity